The Leishmania spp. are kinetoplastid protozoa that are transmitted to humans and other mammalian hosts by, e.g., a sand fly vector. The spectrum of symptomatic human leishmaniasis is wide, and the most important factor determining the clinical outcome of infection seems to be the species of Leishmania. Nonetheless, there are variable clinical presentations of disease due to each species (10, 22, 30), and increasing reports document atypical presentations of leishmaniasis, sometimes but not always in the setting of the immunocompromised (16, 53). Differentiation between the Leishmania species is an issue since there are overlapping and dynamic geographic regions of risk, and different susceptibilities to treatment (7, 15). Thus, a method of diagnosis that is sensitive enough to detect low levels of the parasite in asymptomatic or early symptomatic infection, and can distinguish between the different Leishmania species, would be of tremendous utility in endemic and non-endemic regions (38).
Procedures for diagnosis of leishmaniasis are often invasive, and isolates are frequently difficult to grow in vitro. Tests to distinguish between the Leishmania species have traditionally involved separation of isoenzymes in culture-derived parasites, which takes several weeks (12). The Leishmania spp. have been detected in and isolated from blood cultures of subjects with all forms of leishmaniasis (20, 39, 40), and in the blood of asymptomatic individuals living in regions of risk (11, 25, 34, 39). The possibility that leishmania may be present in the bloodstream more often than previously recognized led us to hypothesize that amplification-based methods to detect parasite DNA in blood or serum might be a feasible means of diagnosis.
Nucleic acid-based methods avoid the need for parasite cultivation, replacing this with either hybridization or amplification (24, 24, 35, 39, 67). The latter approaches provide the advantage of increased sensitivity. Amplification methods reported for the detection of individual Leishmania species include conventional PCR (4, 21, 63) or quantitative PCR methods, including reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), DNA-based qPCR, quantitative nucleic acid sequence-based amplification (QT-NASBA) and in situ hybridization to quantify Leishmania spp. in blood or tissue samples (63).
Protozoa belonging to the order Kinetoplastida, including Leishmania spp. and Trypanosoma spp., are characterized by a prominent kinetoplast structure containing the mitochondrial DNA in the parasites' single mitochondrion. Whereas Leishmania spp. have 35-36 chromosomes in their nuclear genomes (52), the kinetoplast contains hundreds of DNA maxicircles encoding genes that are destined for RNA editing, and thousands of DNA minicircles, circular molecules with a conserved origin of replication encoding guide RNA sequences for RNA editing (13). Because of their abundance, specificity and repetitive nature, kinetoplast DNA (kDNA) sequences have frequently been targeted for nucleic acid based detection (33, 42, 43, 48, 49). A drawback of the use of kDNA for parasite quantification is the uncertainty of whether the kDNA copy number differs between Leishmania species, strains, and growth stages.
The goal of this study was to develop a serial nucleic acid amplification based method for diagnosis and speciation of Leishmania spp. parasites in human or animal-derived tissues. As such we developed a set of primers and probes for serial qPCR assays. The assays were sensitive enough to detect low levels of parasites, and to distinguish between Leishmania species in human specimens. Using non-species discriminating probes, we quantified the relative differences in kinetoplastid DNA (kDNA) copy numbers between parasite species, among isolates of the same species, and between stages of the same parasite strain. The serial qPCR assays has potential applications for diagnosis and species discrimination, as well as novel approaches to determining parasite load and following treatment response in infected humans.
The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established.
We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species, and quantify parasites in clinical or environmental specimens. SYBR green qPCR may be employed, with corresponding Taqman assays for validation. Screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species discriminating probes with melt curve analysis. The assay was sufficient to detect, speciate and quantify Leishmania spp. in sera, cutaneous biopsies, or cultured isolates from subjects in Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kDNA probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6 fold between Leishmania species, but differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and speciation, and for absolute quantification when compared to a standard curve from the same Leishmania species.